【摘要】 目的：本研究旨在开发一种新型纳米材料_hCeO₂@CA-074Me NPs，并系统评估其在炎症条件下优化成骨微环境的分子机制及潜在应用价值。方法：采用模板法合成中空二氧化铈纳米颗粒（_hCeO2 NPs），并通过静电吸附方式负载组织蛋白酶B抑制剂（CA-074Me），制备纳米复合材料_hCeO₂@CA-074Me NPs；通过透射电子显微镜（TEM）、X射线衍射（XRD）、傅里叶变换红外光谱（FTIR）及X射线光电子能谱（XPS）对材料形貌、晶体结构和表面化学性质进行表征；采用CCK-8实验评估纳米颗粒的生物相容性，并利用酶模拟活性试剂盒以及ROS检测试剂盒分析其抗氧化特性；以牙龈卟啉单胞菌脂多糖（P.g-LPS）刺激RAW264.7巨噬细胞构建体外炎症模型，通过RTqPCR和Western blot检测CTSB-NLRP3炎症通路相关基因（CTSB、NLRP3、ASC、Caspase-1、IL-18和IL-1β）及CTSB的蛋白表达变化，同时评估巨噬细胞表型极化标志物（M1:IL-6、TNF-α、IL-1β;M2:Arg、IL-10、TGF-β）的基因表达情况；利用P.g-LPS刺激MC3T3-E1前成骨细胞构建体外炎症性成骨模型，通过ALP染色及活性定量分析、茜素红染色及成骨相关基因（Runx2、Col-1、OPN、OPG）表达分析，探究纳米材料在炎症条件下的成骨促进作用。结果：合成的_hCeO₂@CA-074Me NPs呈空心结构[粒径（198.85±28.39） nm]，具备高Ce3+比例（18.38%）及优异的酶模拟活性，可显著清除细胞内ROS（P<0.05）。该材料可显著下调CTSB-NLRP3通路相关基因和CTSB蛋白表达（P<0.05）并促进巨噬细胞向M2表型极化（上调Arg、IL-10、TGF-β的mRNA表达，P<0.05），有效缓解炎症反应。同时，_hCeO₂@CA-074Me NPs显著提升MC3T3-E1细胞的ALP活性（P<0.05）及矿化能力（茜素红染色面积显著增加，P<0.05），并协同上调Runx2、Col-1、OPN、OPG成骨标志基因表达（P<0.05）。结论：_hCeO₂@CA-074Me NPs通过多靶点抑制炎症信号、重塑免疫微环境并直接促进成骨分化，为炎症性口腔颌面骨缺损的修复提供了一种具有转化潜力的创新纳米治疗策略。更多还原

【Abstract】 Objective: This study aimed to develop a novel nanomaterial,_hCeO₂@CA-074Me NPs, and systematically evaluate its molecular mechanisms and potential therapeutic value in optimizing the osteogenic microenvironment under inflammatory conditions. Methods: Hollow cerium dioxide nanoparticles（_hCeO₂ NPs） were synthesized using a template-assisted method and subsequently loaded with the cathepsin B inhibitor CA-074Me via electrostatic adsorption to prepare the composite nanomaterial_hCeO₂@CA-074Me NPs. The morphology, crystalline structure, and surface chemical properties were characterized by transmission electron microscopy（TEM）, X-ray diffraction（XRD）, Fourier-transform infrared spectroscopy（FTIR）, and X-ray photoelectron spectroscopy（XPS）. The biocompatibility of the nanoparticles was assessed using the CCK-8 assay, and their antioxidant properties were evaluated using enzyme-mimetic activity kits and ROS detection assays. An in vitro inflammatory model was established by stimulating RAW264.7 macrophages with Porphyromonas gingivalis lipopolysaccharide（P. g-LPS）. Expression levels of genes related to the CTSBNLRP3 inflammatory pathway（CTSB, NLRP3, ASC, Caspase-1, IL-18, and IL-1β） and CTSB protein were analyzed via RT-qPCR and Western blot. Additionally, the expression of macrophage polarization markers（M1: IL-6, TNF-α, IL-1β; M2: Arg, IL-10, TGF-β） was evaluated. An inflammatory osteogenesis model was also established by treating MC3T3-E1 pre-osteoblasts with P.g-LPS. Osteogenic activity was assessed using ALP staining and quantification, alizarin red staining, and analysis of osteogenesis-related gene expression（Runx2, Col-1, OPN, OPG）. Results: The synthesized_hCeO₂@CA-074Me NPs exhibited a hollow structure with an average diameter of（198.85 ± 28.39） nm, a high Ce³⁺ ratio（18.38%）, and excellent enzyme-mimetic activity, effectively scavenging intracellular ROS（P<0.05）. These NPs significantly downregulated the expression of genes and proteins related to the CTSB-NLRP3 pathway（P<0.05）, promoted M2 macrophage polarization by upregulating Arg, IL-10, and TGF-β expression（P<0.05）, and effectively alleviated the inflammatory response. Furthermore,_hCeO₂@CA-074Me NPs markedly enhanced ALP activity and mineralization in MC3T3-E1 cells（P<0.05）, along with significant upregulation of osteogenic marker genes Runx2, Col-1, OPN, and OPG（P<0.05）. Conclusions:_hCeO₂@CA-074Me NPs inhibit inflammatory signaling through multiple targets, modulate the immune microenvironment, and directly promote osteogenic differentiation. These findings highlight their potential as an innovative nanotherapeutic strategy for the treatment of inflammation-associated maxillofacial bone defects.更多还原